Tissue factor pathway inhibitor (TFPI) is an essential anticoagulant protein. Decreased plasma TFPI is associated with venous and arterial thrombosis and pharmaceutical agents that block TFPI activity are being developed to treat patients with hemophilia. TFPI is an alternatively spliced protein and all isoforms are capable of inhibiting tissue factor (TF) initiated blood coagulation. During the previous funding period we found that TFPI? is the primary isoform on endothelium, while TFPI? is the primary isoform within platelets. Using murine model systems we also found that hematopoietic cell TFPI, which is primarily platelet TFPI?, limits thrombus growth following vascular injury and weakens the hemostatic response in hemophilia. These results suggested that TFPI? has a specific anticoagulant activity that is not performed by TFPI?. We recognized that the basic C-terminal region of TFPI?, which is not present in TFPI?, and the basic region of the FV B-domain have striking homology and sought to define how TFPI? may specifically interact with FV/FVa. We have now demonstrated that TFPI? effectively inhibits prothrombinase assembled with forms of FVa that retain the acidic region of the FV B-domain, such as that activated by FXa or found within platelets, but not with forms of FVa that have the entire B-domain removed, such as that activated by thrombin. Our preliminary data further demonstrate that the embryonic lethality of TFPI null mice is rescued by breeding into PAR-4 null mice, providing the first demonstration that TFPI activity and platelet function directly counterbalance each other to prevent development of consumptive coagulopathy. These data strongly suggest that TFPI anticoagulant activity is mediated by two separate mechanisms: 1) FXa-dependent inhibition of TF-FVIIa, mediated by Kunitz domains 1 and 2 that are present in all TFPI isoforms; and 2) The inhibition of early forms of prothrombinase that assemble following activation of FV by FXa or release of FVa from collagen-activated platelets. This inhibition requires an exosite interaction between the basic C-terminal region of TFPI? and the acidic region of the B-domain of FV. This proposal is focused on identifying the biochemical interactions that allow for inhibition of prothrombinase by TFPI? and defining its physiological importance. Aim 1 will examine the contribution of a well-conserved LIKT amino acid sequence to the inhibition of prothrombinase by TFPI?. Aim 2 will further characterize the anticoagulant function of platelet TFPI? by transplanting bone marrow expressing different forms of platelet TFPI into irradiated TFPI-/-/PAR4-/- mice using ex vivo studies of platelet activity and in vivo studies of thrombus growth following vascular injury. Aim 3 will investigate TFPI?-mediated inhibition of prothrombinase in murine model systems in which FV/FVa are specifically altered. Inhibition of prothrombinase is a newly recognized anticoagulant mechanism not performed by any other human protein. Therefore, it is anticipated that the results of the proposed experiments will provide new insights into the pathophysiology of a wide range of thrombotic and bleeding disorders and potentially lead to new approaches for their treatment.